Main research topics of the Neisseria unit, National Reference Center for the Meningococci, are in the field of the molecular epidemiology and pathogenesis of infections due to Neisseria meningitidis, including the molecular typing of clinical isolates from invasive infections. Experimental research focuses on the characterization of virulence factors and protective antigens through the development of cellular and murine models.

1. Molecular pathogenesis of meningococcal infections.

Neisseria meningitidis (Nm) is a frequent commensal bacterium of the human nasopharynx. Invasive infection occurs when the bacteria, that first attach to the respiratory epithelium, cross this barrier to invade the blood. They can further cross the blood brain barrier to provoke meningitis. Other localization can be encountered such as, arthritis or pericarditis. The adhesion of Nm to epithelial and endothelial cells is a crucial stage in the infectious process which implies complex interactions between the bacteria and their target cells. These interactions begin with the stage of initial (localized) adhesion and is followed by the stage of intimate adhesion. The protein PilC1 is the major adhesin, and the expression of pilC1 is induced transiently during the initial adhesion. This induction depends on a transcription site localized in the Neisseria contact regulation element (CREN) in the promoter region of pilC1. Another gene, crgA, coding the protein CrgA, a LysR-type transcriptionnal regulator, is also induced by a CREN-dependent process. CrgA modulates the expression of bacterial surface structures involved in adhesion, such as the type IV pili and the capsule, by coordinately regulating the expression of the genes pilC1, pilE, sia et crgA to permit the transition from initial to intimate adhesion.

There is no animal model reproducing the complete infectious and invasive process, from the colonization of the respiratory tract to the invasion of the blood and the meningeal spaces. Based upon epidemiological studies showing a correlation between the incidence of influenza and meningococcal outbreaks, we have developed a mouse model of sequential infection with the influenza A virus (IAV) and N. meningitidis. IAV primary infection induces a transitory phase of susceptibility of mice to the superinfection by N. meningitidis, inducing pneumonia and a subsequent bacteremia. This mouse model of invasive meningococcal infection mimics the main steps of the disease in humans and permits to analyze the functions of known or genome-derived new genes. A capsule defective mutant is rapidly cleared from the lungs, whereas a crgA mutant is invasive. Other experiments show that strains sharing an altered penA gene and expressing diminished susceptibility (PenI) to penicillin have also impaired virulence for mice.

Since penicillin-binding proteins (PBP) are involved in the synthesis of peptidoglycane, we have analyzed the detailed structure of peptidoglycane by mass-spectrometry and chromatography and characterized 28 different muropeptide species. PenI clinical isolates as well as isogenic penA mutants from a susceptible PenS strain have altered structure of their peptidoglycane, represented by an augmentation in GlcNac-MurNac pentapeptide. These structural alterations of peptidoglycane are directly linked to the structural changes of PBP2 in penI strains, suggesting that N. meningitidis isolates with diminished susceptibility to penicillin G have important alterations of their cell wall. Moreover, these alterations may affect the virulence of such strains, as suggested by virulence studies in the mouse model.

This model of meningococcal respiratory challenge reproduces the main stages of the natural meningococcemia in adult mice. Its good reproducibility permits to compare the virulence of various clinical isolates with homologous or different genotype and different isogenic variants of the same parent strain. Therefore, we investigated the crucial question of the correlation between virulence and genotype in invasive strains. This permitted us to demonstrate that meningococcal virulence is directly determined by particular genotypes (clonal complexes) more than the immune specificity of the capsule in terms of its ability to colonize the respiratory epithelium and its invasivity, evaluated from the intensity of the bacteremia. The prominant role of the genotype over the capsular type is confirmed by transformation experiments of the capsule genes. These results are important to consider since the current capsular polysaccharide-based vaccines are aimed to the elimination of epidemic serogroups but may have no impact on the incidence of hypervirulent genotypes. In addition, the problem of the emergence of escape variants to the capsular polysaccharide vaccine-induced immunity, either by serogroup replacement or by capsule switching after transformation and allelic recombination, is still a matter of concern.

Although important advances have been made from full genome sequencing in the identification of putative vaccine candidates, the search for protective antigens against invasive meningococcal infections is often hampered by the genetic variability of most bacterial surface antigens exposed to host immunity. Our studies on penicillin-binding proteins have shown that PBP2 shares N terminal half that are conserved among penicillin susceptible or resistant strains as well as several other conserved domains in the C terminal half Moreover, PBP2 is naturally immunogenic, as assessed from serological conversion observed in patients convalescent from meningococcal disease. We showed experimentally that antibodies to PBP2 are protective against invasive challenge of mice and that vaccination with recombinant PBP2 induced significant protection against bacteremia.

Surveillance of meningococcal infections (MK Taha, JM Alonso).

In industrialized countries, meningococcal diseases occur as sporadic cases due to diverse genotypes, mainly serogroup B, C and W135 isolates. In this context, host immunological defects immunosuppressive conditions or the infection hypervirulent strains are suspected in any invasive infection, whereas most frequently, the infection of the normal host usually leads to asymptomatic carriage and naturally acquired immunity. In the "sub-Saharan African meningitis belt" (from Senegal to Ethiopia), meningococcal meningitis epidemics occur periodically that are due to homogenous genotypes, mainly strains of serogroups A and W135. In these cases, the virulence of the epidemic clone determines the transmissibility and severity of the disease among immunologically naive populations. The national reference centre for meningococci expertises an average of 1,000 strains of N. meningitidis per year, that are sent by 700 national collaborating laboratories. Approximately half of the strains are from invasive infections (meningitis, meningococcemia, arthritis and pericarditis). Sporadic cases occur with an annual incidence below 1 per 100,000 inhabitants. Records from 2005 show that serogroup B remains the most frequent (>60%), followed by serogroup C (26%), serogroup W135 (6%), and serogroup Y (3%), which is mainly isolated from immunocompromised hosts. We pay special attention to the evolution of phenotypes and genotypes of invasive strains, particularly concerning their antigenic structure, their susceptibility to antibiotics, and more generally their sequence type, as assessed by multilocus sequence typing that permits strains from invasive infections, either in France or in Africa, shows a continuous diversification with the emergence of local clones, but without clonal expansion. to determine their belonging to a particular clonal complex. For example, serogroup W135 appeared from 2000 as a new epidemic variant, with the emergence of a majority of strains of the clonal complex ST11. Its detection in epidemic conditions in sub-Saharan Africa in 2001 alerted for the risk of a pandemic spread. Nevertheless, the follow-up of the genotypes of W135